unplugged

Promoter Structure

To identify cis-regulatory regions that are responsible for the normal unpg expression in embryos, six restriction fragments encompassing 20 kb of unpg genomic sequence were tested for their ability to direct expression of a lacZ reporter gene containing a minimal hsp70 promoter. The only region capable of driving a lacZ reporter gene expression similar to the unpg protein distribution is located at the 3' end of the unpg transcription unit. The 2.7 kb fragment gives characteristic unpg-like expression in the CNS and the cerebral and ganglionic branches of the tracheal system during embryogenesis. This fragment also gives unpg-like expression in the cells around the first tracheal pit at the germband extended stage. Thus a 2.7 kb fragment of unpg 3' flanking sequence contains most of the cis-regulatory elements for the normal unpg expression in embryos (Chiang, 1995)

Transcriptional Regulation

unplugged expression occurs in portions of the tracheal system that penetrate the CNS, including the cerebral branch specific to T1. To test the possibility that genes in the BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and in Ubx, abd-A, Abd-B triple mutants. In Ubx mutant embryos additional unpg expression is observed in cells surrounding the tracheal pits of T2 and T3, indicative of a role for Ubx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants of posterior T2 and T3 toward a T1 identity. In abd-A mutants embryos extra patches of unpg-expressing cells around the tracheal pits extend posteriorly to A7, indicating a role for Abd-A in the repression of unpg expression in the abdominal segments. The homeotic gene Abdominal-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triple mutants (Chiang, 1995).

The specific branching defects observed in unplugged mutant embryos prompted an examination of the relationship between unpg and pointed (pnt), a gene encoding a member of the ets family of transcription factors; pnt is expressed in tracheal placodes and in the developing tracheal branches. In the absence of pnt gene function, tracheal cells fail to migrate and branches do not extend to target tissues. In particular, stalling of the ganglionic branches at the ventral oblique musculature in hypomorphic pnt embryos is reminiscent of the most extreme phenotype observed in unpg embryos. To determine the regulatory relationship between pnt and unpg genes, pnt homozygous embryos were immunostained with Unplugged-specific antibody, in order to follow the fate of the ganglionic branches. At stage 12.5, ganglionic branch precursor cells migrating toward the CNS are clearly visible in wild-type embryos. In pnt mutant embryos, no migratory cells that are expressing Unpg protein can be detected; however, a few precursor cells accumulating low levels of Unpg protein occasionally can be identified. By stage 14, the ganglionic branches of normal embryos are well developed, as is evident from a group of 8 to 9 unpg-expressing cells along the ventrolateral region of each hemisegment. In pnt mutant embryos, only 3 to 4 unpg-expressing cells can be detected; these unpg-expressing cells are clustered in a group suggesting that the precursor cells remain immobile and fail to extend from the tracheal pits. These results suggest that the ganglionic branch phenotype in pointed embryos may be in part due to a loss or reduction of unpg gene expression and failure of unpg-expressing cells to extend into the CNS (Chiang, 1995). It would be of interest to determine if unpg functions downstream of pnt in the ventral midline of the CNS.

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